B Cell Development Flashcards
Where do B cells develop?
In the bone marrow
Where do B cells engage Ag?
In the peripheral lympoid organs
How are the different stages of B cell development distinguished?
- rearrangment status of HC and LC genes
- presence cell surface markers such as BCR
Pro-B cell
- pro as is progenitor
- Early stage where the cell becomes commited to becoming a B cell
- recieves signals from the bone marrow stromal cells that promote proliferation and further development
Immature B cell
- Expresses IgM on the cell surface
- HC and LC are rearranged and has BCR expressed
Mature B cell
- Expresses membrane IgM and high levels of IgD on cell surface
- mature B cells circulate amoungst the 2ndary lymphoid organs (some live only days while others circulate for several weeks)
What genes does the Pro-B cell turn off/on
- Turns on B cell genes such as CD19, Ig alpha/beta, pre-BCR, and BLNK. Also turns on transcription factors such as E2A, early B cell factor (EBF) and PAX5
- turns off/represses genes important for other lineages (Ex: Pax5 turns off expression of the T cell lineage transcription factors)
What part of V(D)J recombination happens first and what stage of the B cell does this start?
heavy chain rearrangment starts when the RAG-1/2 genes are turned on in the Pro-B cell
What order does the HC V(D)J recombination happen in the Pro B cell?
Begins D to J followed by V to DJ
What happens when B cells lack RAG-1/2?
They do not get past pro-B cell develpment and have a SKID phenotype
Severe Combined Immunodeficency
- SKID
- Defect in B and T cell function and they have severe impaired adaptive immunityand are severly immunocomprimised
How can people with SKID be treated?
- live in a sterile enviroment
- bone marrow transplant
- gene therapy
How can RAG deficiency be studied and potencial treatments tested?
A mouse model
What makes a mouse model a good model to study human biology?
- reproduce quickly
- ethical reasons
- they are vertebrates and physiologically similar to humans
- have a similar # of genes
- genetic abnormalities that cause disease in humans will often result in similar disease in mice
Transgenic mice
mice with additional DNA (we add DNA)
Knock Out mice
Mice with an altered gene so that it no longer makes a functional protein or mRNA product
Knock in mouse
Mouse with a replaced gene witha copy with a specific mutation
How can RAG deficencies be modeled in a mouse?
Generate a mouse with a knock-out of Rag1 gene
Flow cytometry
Technique to measure the expression of specific proteins on the surface or on the inside of indivisualcells. To do this you stain cells with florescently labelled Ab
Allows us to phenotype cells in a complex mixture
What is forward scatter in flow cytometry?
measurment of cell size (ensure single cells)
What is side scatter in flow cytometry?
measurment of granularity, allows us to remove debris
Pre- B cell
- Precursor B cell
- chracterized by its rearranged HC
- there are 2 stages for this B cell (early/large stage and late/small stage)
- several rounds of proliferation at this stage
What is the defining feature of the pre-B cell stage?
heavy chain expressed and pairs with surrogate LC to form the pre-BCR
Describe what is happening in the genes of the hematopoietic progenitor
- Germ-line configuratiom
- IgH locus contains promoters upstream of each V segment which bind RNA polymerase II (sites where transcription is iniciated)
- enhancer elements in intron between the variable and constant reigons (E mu) and 3’ of alpha constant reigon
- IgH gene is in inactive chromatin in non-B cells including early hematopoietic progenitors (not accessible to RNA polymerase II and transcriptional activators)
Describe what is happening in the genes of the early Pro-B cell
- Chromatin becomes accessible
- B cell specific gene regulatory proteins are expressed and bind to enhancer elements (B cell specific transcription factor, Oct-2, binds promoter)
- not to much transcription from V segment promoters because they are to far from enhancers
- VDJ recom of the HC is happening and the recombination of the J and D segments bring the V segment closer to the enhancers and allows for efficent transcription of the HC
What is the surrogate LC composed of? What cells express pre B cells?
2 proteins (VpreB and lamda5)
All pre-B Cells
How does the Pre-BCR begin signaling? Why does the pre-BCR need to begin signalling?
- the surrogate LC’s, VpreB has extra C-terminal acidic residues and lambda5 has an extra N-terminal basic residues which allows for multiple pre-BCRs to self-aggregate with no ligand and this iniciates signalling
Pre-BCR signalling is a critical developmental checkpoint
X-linked Agannaglobulinemis (XLA)
paitents witha a Block in B cell development at the pre-B cell stage (no mature B cells= no humoral immunity) but T cell development is normal
This is caused by a mutaion in the btk gene on the X chromosome
These paitents are particularly susceptible to extracellular bacterial infections
Who is mainly effected by XLA
Men because they only have 1 X chromosome so most woman with this mutation would be heterozygous (asymtomatic carriers)
How are XLA paitents treated?
Current therapy is directed at reintroducing Ab rather than correcting defect in B cells
- patients recieve weekly gammaglobulin injections (pooled IgG from many donars to restore some humoral immunity)
- these paitents are treated more liberally with antibiotics
When does LC rearrangment start in a B cell?
in the pre-B cell stage
what does the recombination of a functional HC and LC result in?
A IgM+ immature B cell
What happens to IgM+ B cells?
They leave the bone marrow
Why might junctional diversity sometimes cause problems?
- in creating junctional diversity there may be nonsense and frameshift mustations made which can prevent B cell development
How many heavy chain alleles does each B cell have?
2
How many LC alleles does each B cell have?
4 alleles (2 kappa and 2 lambda)
How do VDJ rearrangments occur?
In an ordered progression; HC before LC and kappa LC before the lambda LC
Successful HC rearrangment
One that can make a complete HC protein that can pair with the surrogate LC and form the pre-BCR
After this occurs the LC can now be rearranged
What happens from the self-aggregation/cross-linking induced signaling in an early/large pre-B cell? Then, late/small pre-B cell?
Early/large pre-B cell:
1. survival proliferation
2. Decrease in Rag1/2 and decrease in VDJ rearrangment (HC allelic exclusion)
late/small pre-B cell:
3. decrease in pre-BCR and IL-7 signals and decreased cell proliferation
4. increase in Rag 1/2 activity
5. Increase LC VDJ rearrangment
Describe theVDJ rearrangment in the HC?
D to J recom first occurs in both chromosomes and is almost always successful
V to DJ occurs in 1 chromosome first and has a 1/3 chance of being successful (if it fails 3 times the cell will die)
Describe the VDJ rearrangment in the LC
V to J rearrangment starts at 1 allele of the kappa gene and moves to the lambda after 2 kappa chains tried (has 4 chances for a successful rearrangment)
**If you have a functional heavy chain the likelyhood for a successful LC rearrangment increases
How does the BCR emmit signals after VDJ rearrangment is done? What does this do?
BCR’s can do ligand independent signalling which means that just having receptors on the surface makes enough of a signal to stop LC rearrangment
This signal turns off Rag genes and limit access to other LC loci
Allelic exclusion
Functional rearrangment of 1 HC/LC allele prevents the functional rearrangment of the other alleles
What does allelic exclusion ensure?
That each mature B lymphocyte bears a BCR of 1 specificity (only 1 HC protein and 1LC protein will be expressed)
How can we show allelic exclusion experimentally?
The addition of functionally rearranged HC and LC genes in a mouse should prevent the endogenous HC and LC from rearranging and expressing on the surface
(use transgenic mice)
Lysozyme
anti-bacterial protein (gram +) that is found in human saliva and tears aswell as chicken egg whites
HEL
Hen Egg Lysozyme, a well studied protein that have Ab that recodnizes it
How do you make a HEL transgenic mouse?
You obtain genomic DNAs containing rearranged HC and LC genes that encode for an AB that recodnizes HEL. The heavy constant reigons for IgM and IgD and endogenous regulatory elements need to be included so the BCR will develop normally
Then you use the genomic DNA to create a transgenic mouse. The transgenes are present in all cells but only expressed in B cell lineages and are randomly inserted into the genome so that the endogenous IgH and IgL loci are unaffected
Allotypes (give example)
Minor sequence differences in HC/LC constant reigons (alleles); this does not mean they are functionally different
For the transgenic mouse the transgenic BCR HC is an a allotype and the endogenouse BCR HC iis the b allotype
Transitional B cells
- immature B cells that have left the bone marrow (takes about 1-2 weeks to get out of the bone marrow)
- transitioning between immature and mature stages
- this stage has recieved a signal from cells in the spleen to continue development
- B cell activating factor (BAFF) is required for survival and maturation
BAFF
B cell activating factor
Secreted by cells in the spleen and promotes the survival and continued development of B cells and is required for mature B cell survival
Describe the expression of mIgM, mIgD, and BAFF receptors on T1, T2, and mature B-2 cells.
T1: IgM high, IgD none to low, BAFF receptors can be expressed but not all the time
T2: IgM high, IgD intermediate, BAFF receptors present
Mature B-2: IgM intermediate, IgD high, BAFF receptors present
How long does it take for the transition to happen from a transitional and mature B cell?
about 5 days
How is the expression of isotypes other than IgM and IgD mediated?
by DNA recombination at switch reigons
How are constant reigons orientated in regards to the V heavy chain
constant reigons are 3’ to VH
How are IgM and IgD transcribed?
Together (as 1 primary RNA transcript) and mature IgM and IgD are generated depending on where transcript is cleaved and polyadenylated (addition of nucleotides)
How is is that immature B cells only express IgM when they are normally transcribedas 1 primary RNA transcript?
immature cells only cleave at Poly-A site 2 and splice to the mu constant reigon exons (delta reigon that encodes IgM gets cleaved and not transcribed)
Where does the mu and delta gene reigons get cut in mature B cells?
They preferencially cut at Poly-A site 4 and splice to the mu constant reigon but can also use Poly-A site 2
Negative selection
the removal of self-reactive B cells resulting in the B cell repertoire being tolerant of self antigens
Central tolerance
removal of self-reactive lymphocytes in primary lymphoid organs (B cells in bone marrow and T cells in the thymus)
What are 3 ways that the body removes B cells that recodnize self-antigens?
- colonal deletion
- anergy (functional unresponsiveness)
- recepto editing
Where does negative selection occur?
In the central and peripherary lymphatic organs
How do B cells decide wheather to mount an immune response or become tolerant?
- B cells need multiple signals to occur in order for them to become activated
- When the B cells see the Ag during B cell development
Colonal Deletion
Getting rid of self-reactive cells by having them undergo programmed cell death (primarily apoptosis)
Why is phagocytosis important for colonal deletion of self reactive B cells?
To prevent more signalling from the B cell fragments (stops DAMP signalling)
Functional anergy
- self reactive cells become non-responsive to Ag (have impaired signalling when AG binds BCR)
- have a shorter half life
- are phenotypically different
What determines wheather a self-reactive cell undergoes colonal deletion?
Multivalent self Ag induce a strong BCR signal when many BCRs are simutaneously engaged on the cell surface
What determines wheather a self-reactive cell undergoes anergy?
Monovalent self Ag bind BCR which engages fewer BCRs on the cell surface and induce a weaker BCR signal
How can colonal deletion and anergy be studied?
Usig the anti-HEL BCR transgenic to study Negative Selection
You can use a anti-HEL BCR transgenic mouse
Why would using a BCR transgenic be useful to study negative selection?
All B cells are of the same specificity and you know the Ag specificity of the transgenic BCR
Receptor Editing
Light chain gene can undergo further rearrangment in self-reactive cells which is a mechanism to rescue self-reactive immature B cells
The B cells arrest in development and starts rearranging LC again (only has a finite time to do this)
B-2 B cells
General B cells that we have talked about throughout all of the lectures
aka: follicular B cell
What are 3 types of nonconventional B cells?
- B-1 B cells
- maginal zone B cells (good at responding to blood borne pathogens in spleen)
- IL-10 secreting regulatory B cells (important for turning off the immune response
Compare B-2 B cells to B-1 B cells.
- major sites of mature cells
- source of cells in adults
- V reigon diversity
- requirement for T cell help
- isotypes produced
- memory
- somatic hypermutation
- Ag recognized
B-2 B cells:
- major sites of mature cells
secondary lymphoid organs
- source of cells in adults
progenitors in the bone marrow
- V reigon diversity
highly diverse
- requirement for T cell help
yes
- isotypes produced
varied
- memory
yes
- somatic hypermutation
extensive
- Ag recognized
varied
B-1 B cells:
- major sites of mature cells
peritoneal and pleural cavities
- source of cells in adults
self renewal of exsiting B-1 cells
- V reigon diversity
limited
- requirement for T cell help
no
- isotypes produced
primarly IgM
- memory
limited
- somatic hypermutation
some
- Ag recognized
primarily carbohydrate and lipid (innate B cells and can make IgM very quickly against bacterial carb and lipid structures)
